The method defined above is employed in chromatographic procedures utilizing monolithic matrices or particle matrices in form of packed or fluidised beds, and also in batch-wise procedures. The purpose of the procedures may be to purify a substance carrying a negative charge, in which case the target substance is bound to the matrix, and, if necessary, further purified subsequent to desorption from the matrix. Another purpose is to remove an undesired substance that carries a negative charge from a liquid. In this latter case, the liquid may be further processed after having been contacted with the matrix in step (i). In both cases and if so desired, the matrix may be reused after desorption of the bound substance.
Other uses are assay procedures involving determination of either the substance carrying the negative charge or of a substance remaining in liquid I.
In previous anion-exchange adsorptions, the positively charged ligands typically have comprised nitrogen structures, such as primary, secondary, tertiary or quaternary ammonium structures. In some instances the ligands had a dual or bimodal functionality by comprising both a charged structure and a hydrophobic structure which has required modifications of the desorption protocols.    Simmonds et al (Biochem. J. 157 (1976) 153-159); Burton et al (J. Chromatog. A 814 (1998) 71-81); and Yon et al (Biochem. J. 151 (1975) 281-290) have described anion exchange ligands that comprise saturated hydrocarbon groups.    Crowther et al (J. Chrom. 282 (1983) 619-628); Crowther et al (Chromatographia 16 (1982) 349-353); Wongyai (Chromatographia 38(7/8) (1994) 485-490); Bischoff et al (J. Chrom. 270 (1983) 117-126) have described high pressure liquid chromatography of oligonucleotides and small molecules on reverse phases carrying anion exchange ligands in which there is an aromatic component.
See also Sasaki et al (J. Biochem. 86 (1979) 1537-1548) in which a similar effect from an anion-exchanger based on a hydrophobic matrix is discussed.
Serine proteases have been affinity adsorbed/desorbed to/from matrices to which p-aminobenzamidine has been covalently linked via the para amino group. See    Chang et al (J. Chem. Tech. Biotechnol. 59 (1994) 133-139) who used an adsorption buffer in which the pH is higher and the salt concentration is lower than in the desorption buffer;    Lee et al (J. Chromatog. A 704 (1995) 307-314) who changed the pHs in the same manner as Chang et at but without change in salt concentration; and    Khamlichi et al., J. Chromatog. 510 (1990) 123-132 who used ligand analogues for desorption. The pH-values during adsorption and desorption were the same. Desorption by only increasing the ionic strength failed.
None of the methodologies in these three articles describe successful desorption processes under anion-exchange conditions.
WO 9729825 (Amersham Pharmacia Biotech AB) discloses mixed mode anion-exchangers providing interactions based on charges and hydrogen-bonding involving oxygen and amino nitrogen on 2-3 carbons' distance from positively is charged amine nitrogen. The publication is based on the discovery that this kind of ligands can give anion-exchangers that require relatively high ionic strengths for eluting bound substances.
WO 9965607 (Amersham Pharmacia Biotech AB) discloses cation-exchangers in which there are mixed mode ligands that require relatively high ionic strengths for eluting bound substances.
WO 9729825 (U.S. Pat. No. 6,090,288) and WO 9965607, which give anion and cation exchange ligands, respectively, that require relatively high elution ionic strength are incorporated by reference.
WO 9808603 (Upfront Chromatography) discloses separation media of the general structure M-SP1-L where M is a support matrix that may be hydrophilic, SP1 is a spacer and L comprises a mono- or bicyclic homoaromatic or heteroaromatic moiety that may be substituted (a homoaromatic moiety comprises an aromatic ring formed only by carbon atoms). The substituents are primarily acidic. The separation medium is suggested for the adsorption of proteins, in particular immunoglobulins, by hydrophobic interactions rather than ion-exchange (salt concentration up to 2 M).
WO 9600735, WO 9609116 and U.S. Pat. No. 5,652,348 (Burton et al) disclose separation media based on hydrophobic interaction. Adsorption and desorption are supported by increasing or decreasing, respectively, the salt concentration of the liquid or changing the charge on the ligand and/or the substance to be adsorbed/desorbed by changing pH. The ligands typically comprise a hydrophobic part that may comprise aromatic structure. Some of the ligands may in addition also contain a chargeable structure for permitting alteration of the hydrophobic/hydrophilic balance of the media by a pH change. The chargeable structure may be an amine group.
U.S. Pat. No. 5,789,578 (Burton et al) suggests to immobilise a thiol containing ligand, such as 3-mercaptopropionic acid, gluthathione etc, by addition of the thiol group over carbon-carbon double bond attached to a support matrix. The inventors in this case neither employ nor suggest the use of the material obtained for anion-exchange adsorptions.
Dipolar adsorbents prepared by coupling sulphanilic acid using epichlorohydrin has been described (ligand+spacer=—CH2CHOHCH2N+H2C6H4SO3−) (Porat et al., J. Chromatog. 51 (1970) 479-489; and Ohkubo et al., J. Chromatog. A, 779 (1997), 113-122). The articles do not disclose a separation method in which the ligand is positively, and the substance to be removed negatively, charged.
WPI Abstract Accession No. 86-312313 (=DD-A-237844, Behrend et al) describes the use of 2,4,6-trihalo-1,3,5-triazine for binding substances RHNR′X to carriers inter alia to cellulose. R is hydrogen, aryl or alkyl. R′ alkylene or arylene. X is carboxy, sulphonyl, phosphate, phosphonate, boronate, etc.